Influence Of Period Gene Knocking-out On The Antibacterial Ability Of Bombyx Mori

Circadian clock,a physiological mechanism that controls the spatiotemporal rhythm,affects a variety of biological functions.Bombyx mori is an invertebrate model insect of the Lepidoptera,has high degree of domestication and economic value.The core members of the Bombyx mori circadian clock loop are quite different from mammals and classic model insects such as Drosophila melanogaster and Caenorhabditis elegans in terms of protein molecular evolution and interaction networks.Analysing the molecular mechanism of circadian clock in Bombyx mori is important for understanding the molecular mechanism of circadian clock action of Lepidopteran insects,Bombyx mori molecular breeding and Lepidopteran pest control.In order to explore the relationship between Bombyx mori circadian clock and innate immune system,we used the Period knock-out mutant(Per-/-)to reveal the impact of the Per gene knock-out on the viability of the Bombyx mori in high temperature stress and bacterial infection.The main results are summarized as follows:(1)Transcription and protein level detection of Per knock-out mutantsThe phylogenetic tree was constructed using 40 different PER proteins,and it was found that the Bombyx mori PER protein has the highest homology with Manduca sexta(～90.00%),and also has high homology with Diptera insects such as Drosophila melanogaster(～70.00%),but the homology with Notochords animal is only about 60.00%.The structure of PER is highly conserved,with two PAS motifs and one PAC motif at N-terminal.The 3D structure shows that Bombyx mori PER shares high similarity with human PER protein(49.00%).The Bombyx mori genome contains a single copy of Per gene,implying that the one PER may be responsible for the complex functions of mammals PER1/PER2/PER3.Based on the structural characteristics of Bombyx mori Per,the Per knock-out mutant(Per-/-)was identified.Due to sequence insertion and point mutation,the Per gene of the mutant introduced a stop codon in the coding region,which resulted in the early termination of transcription and the deletion of the translated protein.(2)Knocking out Per does not change the viability of the Bombyx mori under normal feeding conditions,but reduces the viability under high temperature and improves the viability after bacterial infection.Under normal feeding conditions,Per knock-out mutant showed a change in appetite and digestibility compared with WT.There was no significant difference in growth and development between the knock-out mutant and WT during the 5th instar,as well as the viability.However,the T50 of knock-out mutant under high temperature rearing was 25.00%shorter than WT.Oral infection of E.coli and S aureus at the age of 5th instar,there was no significant difference in viability between the knock-out mutant and WT.However,after the injection of E.coli and S.aureus,the T50 of the knock-out mutant was 11.11%and 25.00%longer than the WT respectively.At 18 h after injection,the bacterial load in hemolymph of the mutant was only 40.00%and 14.00%of the WT respectively.Per knock-out mutant showed strong resistance and clearance ability to E.coli and S.aureus(3)Knocking out Per improves the antibacterial peptide gene expression and ROS accumulationThe phagocytic ability of hemocyte showed that there was no significant difference between Per knock-out mutant and WT,but the expression patterns of ActinA1,Ced6,TetraspaninE of the 5th instar mutant were reversed or alternately increased compared with the WT.Although the expression pattern of PPO gene in the knock-out mutant was changed compared with the WT after bacterial infection,there was no significant difference in melanin level and hemolymph melanization ability between mutant and WT.The expression levels of Enbocin and Moricin were significantly up-regulated in the knock-out mutant compared with the WT after bacterial infection.The activation level of the Toll signaling pathway is also significantly higher than the wild type,but the pattern between the two bacteria is different.Moreover,the ROS content in hemocyte of mutants was significantly higher than WT.Knocking out the Per gene of Bombyx mori,although it affects the expression patterns of phagocytosis-related genes and melanization-related genes in hemocyte But it does not directly affect the ability of the circulating blood system to remove bacteria from Bombyx mori after infection.The knock-out mutant relies on the highly activated Toll pathway after infection and the rapid and efficient up-regulation of antibacterial peptide gene expression,which enhances the antibacterial ability of hemolymph through antibacterial peptides,improves the bactericidal ability of hemocyte through ROS,and finally achieves the elimination of the bacteria Conclusion:The knock-out of Per reduces the viability under high temperature environment,but improves the antibacterial peptide gene expression and ROS accumulation to eliminate bacteria effectively,and improves the resistance to bacteria in hemolymph.